AU606453B2 - Recombinant eukaryotic cells production interleukin-2, process and vectors for their preparation and process for the preparation of interleukin-2 - Google Patents
Recombinant eukaryotic cells production interleukin-2, process and vectors for their preparation and process for the preparation of interleukin-2 Download PDFInfo
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- AU606453B2 AU606453B2 AU21796/88A AU2179688A AU606453B2 AU 606453 B2 AU606453 B2 AU 606453B2 AU 21796/88 A AU21796/88 A AU 21796/88A AU 2179688 A AU2179688 A AU 2179688A AU 606453 B2 AU606453 B2 AU 606453B2
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- 108010002350 Interleukin-2 Proteins 0.000 title claims description 82
- 102000000588 Interleukin-2 Human genes 0.000 title claims description 82
- 239000013598 vector Substances 0.000 title claims description 38
- 238000000034 method Methods 0.000 title claims description 31
- 210000003527 eukaryotic cell Anatomy 0.000 title claims description 26
- 238000002360 preparation method Methods 0.000 title claims description 23
- 230000008569 process Effects 0.000 title claims description 18
- 238000004519 manufacturing process Methods 0.000 title description 9
- 239000013612 plasmid Substances 0.000 claims description 75
- 239000002243 precursor Substances 0.000 claims description 51
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 49
- 210000004027 cell Anatomy 0.000 claims description 49
- 230000014509 gene expression Effects 0.000 claims description 33
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 31
- 102000002265 Human Growth Hormone Human genes 0.000 claims description 11
- 108010000521 Human Growth Hormone Proteins 0.000 claims description 11
- 239000000854 Human Growth Hormone Substances 0.000 claims description 11
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 claims description 11
- 229960000485 methotrexate Drugs 0.000 claims description 10
- 108010022394 Threonine synthase Proteins 0.000 claims description 9
- 210000004978 chinese hamster ovary cell Anatomy 0.000 claims description 9
- 102000004419 dihydrofolate reductase Human genes 0.000 claims description 9
- 239000013604 expression vector Substances 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 108010071185 leucyl-alanine Proteins 0.000 claims 4
- QJCKNLPMTPXXEM-AUTRQRHGSA-N Glu-Glu-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CCC(O)=O QJCKNLPMTPXXEM-AUTRQRHGSA-N 0.000 claims 3
- IVGJYOOGJLFKQE-AVGNSLFASA-N Glu-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)O)N IVGJYOOGJLFKQE-AVGNSLFASA-N 0.000 claims 3
- JKGHDYGZRDWHGA-SRVKXCTJSA-N Leu-Asn-Leu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O JKGHDYGZRDWHGA-SRVKXCTJSA-N 0.000 claims 3
- RMODQFBNDDENCP-IHRRRGAJSA-N Pro-Lys-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O RMODQFBNDDENCP-IHRRRGAJSA-N 0.000 claims 3
- 108010025306 histidylleucine Proteins 0.000 claims 3
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- 108010036320 valylleucine Proteins 0.000 claims 3
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- PTSDPWIHOYMRGR-UGYAYLCHSA-N Asn-Ile-Asn Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(O)=O PTSDPWIHOYMRGR-UGYAYLCHSA-N 0.000 claims 2
- LYSHSHHDBVKJRN-JBDRJPRFSA-N Cys-Ile-Ala Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C)C(=O)O)NC(=O)[C@H](CS)N LYSHSHHDBVKJRN-JBDRJPRFSA-N 0.000 claims 2
- XLLSMEFANRROJE-GUBZILKMSA-N Cys-Leu-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CS)N XLLSMEFANRROJE-GUBZILKMSA-N 0.000 claims 2
- 108010054576 Deoxyribonuclease EcoRI Proteins 0.000 claims 2
- DOMHVQBSRJNNKD-ZPFDUUQYSA-N Gln-Met-Ile Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O DOMHVQBSRJNNKD-ZPFDUUQYSA-N 0.000 claims 2
- MUSGDMDGNGXULI-DCAQKATOSA-N Glu-Glu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CCC(O)=O MUSGDMDGNGXULI-DCAQKATOSA-N 0.000 claims 2
- DVLZZEPUNFEUBW-AVGNSLFASA-N Glu-His-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CCC(=O)O)N DVLZZEPUNFEUBW-AVGNSLFASA-N 0.000 claims 2
- YOBGUCWZPXJHTN-BQBZGAKWSA-N Gly-Ser-Arg Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCN=C(N)N YOBGUCWZPXJHTN-BQBZGAKWSA-N 0.000 claims 2
- SENJXOPIZNYLHU-UHFFFAOYSA-N L-leucyl-L-arginine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CCCN=C(N)N SENJXOPIZNYLHU-UHFFFAOYSA-N 0.000 claims 2
- OIARJGNVARWKFP-YUMQZZPRSA-N Leu-Asn-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O OIARJGNVARWKFP-YUMQZZPRSA-N 0.000 claims 2
- QVFGXCVIXXBFHO-AVGNSLFASA-N Leu-Glu-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O QVFGXCVIXXBFHO-AVGNSLFASA-N 0.000 claims 2
- RXGLHDWAZQECBI-SRVKXCTJSA-N Leu-Leu-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O RXGLHDWAZQECBI-SRVKXCTJSA-N 0.000 claims 2
- XOWMDXHFSBCAKQ-SRVKXCTJSA-N Leu-Ser-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC(C)C XOWMDXHFSBCAKQ-SRVKXCTJSA-N 0.000 claims 2
- YVSHZSUKQHNDHD-KKUMJFAQSA-N Lys-Asn-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCCCN)N YVSHZSUKQHNDHD-KKUMJFAQSA-N 0.000 claims 2
- FHIAJWBDZVHLAH-YUMQZZPRSA-N Lys-Gly-Ser Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O FHIAJWBDZVHLAH-YUMQZZPRSA-N 0.000 claims 2
- HYSVGEAWTGPMOA-IHRRRGAJSA-N Lys-Pro-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O HYSVGEAWTGPMOA-IHRRRGAJSA-N 0.000 claims 2
- JHKXZYLNVJRAAJ-WDSKDSINSA-N Met-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(O)=O JHKXZYLNVJRAAJ-WDSKDSINSA-N 0.000 claims 2
- KLXQWABNAWDRAY-ACRUOGEOSA-N Phe-Lys-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 KLXQWABNAWDRAY-ACRUOGEOSA-N 0.000 claims 2
- VHDNDCPMHQMXIR-IHRRRGAJSA-N Phe-Met-Cys Chemical compound SC[C@@H](C(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@@H](N)CC1=CC=CC=C1 VHDNDCPMHQMXIR-IHRRRGAJSA-N 0.000 claims 2
- SSSFPISOZOLQNP-GUBZILKMSA-N Pro-Arg-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O SSSFPISOZOLQNP-GUBZILKMSA-N 0.000 claims 2
- MGJLBZFUXUGMML-VOAKCMCISA-N Thr-Lys-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)O)N)O MGJLBZFUXUGMML-VOAKCMCISA-N 0.000 claims 2
- GYBVHTWOQJMYAM-HRCADAONSA-N Tyr-Met-Pro Chemical compound CSCC[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N GYBVHTWOQJMYAM-HRCADAONSA-N 0.000 claims 2
- FOADDSDHGRFUOC-DZKIICNBSA-N Val-Glu-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N FOADDSDHGRFUOC-DZKIICNBSA-N 0.000 claims 2
- DJQIUOKSNRBTSV-CYDGBPFRSA-N Val-Ile-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](C(C)C)N DJQIUOKSNRBTSV-CYDGBPFRSA-N 0.000 claims 2
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 claims 2
- FSXRLASFHBWESK-UHFFFAOYSA-N dipeptide phenylalanyl-tyrosine Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FSXRLASFHBWESK-UHFFFAOYSA-N 0.000 claims 2
- 108010055341 glutamyl-glutamic acid Proteins 0.000 claims 2
- 108010092114 histidylphenylalanine Proteins 0.000 claims 2
- 108010000761 leucylarginine Proteins 0.000 claims 2
- 108010073101 phenylalanylleucine Proteins 0.000 claims 2
- CNKBMTKICGGSCQ-ACRUOGEOSA-N (2S)-2-[[(2S)-2-[[(2S)-2,6-diamino-1-oxohexyl]amino]-1-oxo-3-phenylpropyl]amino]-3-(4-hydroxyphenyl)propanoic acid Chemical compound C([C@H](NC(=O)[C@@H](N)CCCCN)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 CNKBMTKICGGSCQ-ACRUOGEOSA-N 0.000 claims 1
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
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Description
111111.2511 ZAXMAnsj bdo u w p!qja6p ZAXMAfl1s~doNW1N rIHOrna09V ]d 0112 1.4 COMMONWEALTH OF AUSTRALIA Patents Act 1952 CO0M PL E TE S P E. C I FI-_C>A T I 0ON
(ORIGINAL)
'(CCC'
C C At 0 CI (0 0 00 0 0 1 o 00 0 (1 01 C O CC 0011 0 00 00 0 t CCC C' Application Number Lodged Complete Specification Lodged: Accepted: Published: Lhjs document contains the amendmients, a'!owed ud.; -;'cc,,ion 33 by the Supcrvising Exariitwr on and is correct for printing Priority :1 September 1987 Related Art Name of Applicant Address of Applicant Actual Inventor/s Address for Service
:SANOFI
:40, avenue George V, 75008 Paris, France :Johannes Lupker Brigitte Miloux Willem Roskam F.B. RICE CO., Patent Attorneys, 28A Montague Street, Balmain N.S.W. 2041 Complete Specification for the invention entitled: Recombinant eukaryotic cells production interleukin-2, process arnd vectors for their preparation and process for the preparation of interleukin-2 The following statement is a full description of this invention including -the best method of performing it known to us:-
I.
la The present invention relates to.recombinant Qr to e- s=cSoc -s se-cgoS-yea- %Xtb"zcX,.AL-eit *%ni-c'e>s.
eukaryotic cells producing interleukin-2 It further relates to a process and the vectors for the preparation of these cells. Finally, it further relates to the preparation of interleukin-2 by culture of the said cells.
0 00 oo o o0 0000 00 00 0 0oo 00 0 0 o 0 0 0 0o 0 oo 000 0 00 0 0 0 0 00 0000 0 0 o a The eukaryotic cells according to the invention contain, with the means necessary for their expression, a DNA sequence coding for dihydrofolate reductase and a DNA sequence coding for a hybrid precurso- of interleukin- 2, the signal peptide of which is that of one of the 15 natural precursors of human growth hormone.
These recombinant eukaryotic cells are obtained by a process consisting in transfecting eukaryotic cells with the aid-of a vector which carries, with the means necessary for their expression, a DNA sequence 20 coding for dihydrofolate reductase and a DNA sequence coding for a hybrid precursor of interleukin-2, the signal peptide of which is that of one of the natural precursors of human growth hormone, and in selecting the transfected cells, producing interleukin-2, by growth 25 in successive media, each of which contains a higher concentration of methotrexate than the previous one.
Interleukin-2 is a lymphokine: it is secreted by the mature T lymphocytes of mammals in response to activation by an antigen or a mitogenic compound. It has an essential role by acting on the proliferation and differentiation of different types of cells involved in immune responses Robb (1984), Immunol. Today, 203-209).
Interleukin-2 of human origin has been studied more particularly. It is known to be a protein of (NB l or teclaredat Paris B.A. ou Capitol do 1.403.167.3OaR PLE avenue George VA ANE 7.5093 P 1 J, SIG -r Thv Con i s oncr. V4&ent SIG this 23rd ASE ADD NAM D TITLE OF PE NING, LEGIBLY, iE
RSON
Directet Juridique
I
tIRENE 732 659 332 RC Parts A- 4' 2- 0 0 0 0 0 a o 0 o O 00W Q.0.
0 00 000 o o o 0 o 0 0 O 00 0 133 amino acids which carries a tetrasaccharide attached to the threonine residue in the 3-position Conradt et al. (1986), Carbohydr. Res., 149, 443-450). The mature T lymphocytes synthesize it initially in the form of a precursor of 153 amino acids and secrete it, after elimination of the signal peptide of 20 amino acids by cleavage at the endoplasmic reticulum, and then after glycosylation in the Golgi apparatus, in the form of a protein of 133 amino acids which is called glycosylated mature protein.
The biological properties of interleukin-2 of human origin make it suitable for use as an active principle in drugs useful for the treatment of diseases such as cancers and certain infectious or parasitic 15 diseases. For this use, it seems to be preferable for the interleukin-2 to be in a glycosylated form: the saccharidic side-chain in fact seems to help stabilize the protein and improve its tolerance when administered to patients.
20 The literature contains descriptions of the preparation of interleukin-2 either from healthy peripheral lymphocytes Kniep et al. (1984), Eur. J. Biochem., 143, 199-203) or from a lymphoblastoid cell line such as the Jurkat line Robb et al. (1983), Proc. Natl.
Acad. Sci. USA, 80, 5990-5994).
The methods used have proved rather unsuitable both because of difficulties associated with the culture of healthy lymphocytes and because of the need for an induction phase.
The cloning of a complementary DNA coding for interleukin-2 Taniguchi et al. (1983), Nature, 302, 305-310) was followed by a description of the use of microorganisms rendered suitable for the production of interleukin-2 by means of genetic engineering techniques.
European patent application A-0 089 062 shows the 1 ii
II
i ,i; u i .r i ii
I
i f ;i i! i ii..i 3 3 possibility of using COS monkey cells as well as the bacterium Escherichia coli, which is incapable of glycosylation. European patent application A-0 172 619 discloses the use of various other animal cells, including Chinese hamster ovary cells (CHO cells) in particular.
It is also known that, in order to make it possible, on completion of a transfection, to demonstrate the presence, within a population of eukaryotic cells, of those cells which have actually integrated a a particular vector, it is useful for the said vector to o. carry a DNA sequence whose expression can give the oo transfected cells a selective advantage.
A particularly suitable DNA sequence is a 0 0""0 15 sequence coding for dihydrofolate reductase (this *oo enzyme being abbreviated to dhfr hereafter); described by Subramani et al. ((1981), Mol. Cel. Biol., 854-864), such a sequence, carried by an expression vector, makes 0 00 oo' it possible, after the transfection of cells incapable oo o 0 0 20 of synthesizing dhfr in a functional form (DHFR- cells), to grow, on a medium deficient in hypoxanthene, glycine 0 S and thymidine, only those cells which have actually incorporated the vector.
The interest attaching to the use of a vector 25 which carries, with the means necessary for its expression, a DNA sequence coding for dhfr is strengthened by the fact that, whether the transfected eukaryotic cell is capable (DHFR+ cell) or incapable (DHFR- cell) of synthesizing dhfr in a functional form, it can be the origin of an amplification process leading to an increased production of a protein of interest coded by a DNA sequence which is carried, with the means necessary for its expression, by the said vector. The mechanism of this amplification is yet to be specified. It is known that dhfr is inhibited by the compound known as
I
R: 1 4 methotrexate (L-N-(4-((2,4-diaminopteridin-6-yl)methyl)methylamino)benzoyl)glutamic acid), that the presence of methotrexate in a selective culture medium kills most of the cells and that the only cells to survive are those which have become capable of synthesizing substantial amounts of dhfr. It has moreover been found Kaufman et al. (1982), J. Mol. Biol., 159, 601-621) that, in cells which have incorporated a vector which carries, with the means necessary for their expression, a DNA sequence coding for dhfr and a DNA Sa sequence coding for another protein, this amplified oo0000 ooa production of dhfr is accompanied by an amplified 00 0 0o production of the said protein.
Having constructed vectors which simultaneously 0 o o 15 carry, with the means necessary for their expression, 00 0 0 a DNA sequence coding for a natural precursor of interleukin-2 and a DNA sequence coding for dhfr, the 000 Applicant Company has found that, in the culture of o° 00 eukaryotic cells (and especially CHO cells) which have 20 incorporated one of the vectors in question, such vectors only make it possible under conditions of 0000 ~o0 transient expression, i.e. before the selection of a highly productive line by culture in successive media, each of which contains a higher concentration of methotrexate than the previous one to collect, from the culture medium, smaller amounts of interleukin-2 than those collected from the culture medium of the cells of the same type which have incorporated a vector differing only in the absence of the DNA sequence coding for dhfr and of the means necessary for the expression of this sequence.
These results, although encouraging in terms of the amounts of interleukin-2 which can be obtained after the culture of healthy peripheral lymphocytes or lymphoblastoid cells, show that, in contrast to the observations :s j 5 which could be made with other proteins, such as the surface antigen of the hepatitis B virus or human growth hormone, it is not possible, for the preparation of interleukin-2, to derive all the expected advantage from the use of a DNA sequence coding for dhfr, accompanied by the means necessary for its expression.
Continuing its investigations, the Applicant Company has found, very surprisingly, that, on the first vectors tested, the replacement in the region of the DNA sequence coding for the precursor of interleukin-2 o of the part coding for its signal peptide with a sequence .000 coding for the signal peptide of one of the natural preo 0 cursors of human growth hormone (this protein being abbreviated to hGH hereafter) makes it possible signi- .oo 15 ficantly to improve the level of secretion and even, 0 o 0 with certain constructions, to achieve the expected I a 0a initial level. The Applicant Company is thus providing a solution which, in satisfying the criteria relating o°o 0 thereto, makes it possible to envisage the production 0 0o 0 20 of interleukin-2 on the industrial scale.
According to a first aspect, the invention in S fact relates to recombinant eukaryotic cells producing interleukin-2 which contain, with the means necessary for their expression, a DNA sequence coding for dihydro- 0 25 folate reductase and a DNA sequence coding for a hybrid precursor of interleukin-2, the signal peptide of which is that of one of the natural precursors of human growth hormone.
The invention further relates to a process for .1 30 the preparation of the said cells which consists in 1) transfecting eukaryotic cells with the aid of a vector which simultaneously carries, with the means necessary for their expression, a DNA sequence coding for dihydrofolate reductase and a DNA sequence coding for a hybrid precursor of interleukin-2, the signal S. 1 i C. i 6 peptide of which is that of one of the natural precursors of human growth hormone, and then in 2) selecting the transfected cells, producing interleukin-2, by culture in successive media, each of which contains a higher concentration of methotrexate that the previous one.
Finally, the invention relates to a process for the preparation of interleukin-2 which consists in culturing eukaryotic cells producing interleukin-2, in collecting the culture medium and in separating the interleukin-2 from the other constituents of the said medium, in which process the cultured cells are derived 0 00 ooe from eukaryotic cells transfected with the aid of a 00 vector which simultaneously carries, with the means 0 S00 necessary for their expression, a DNA sequence coding "o00oo 15 for dihydrofolate reductase and a DNA sequence coding 00 0 0 o for a hybrid precursor of interleukin-2, the signal peptide of which is that of one of the natural precursors of human growth hormone, and then selected by 0 00 0 0o culture in successive media, each of which contains a 0 00 0 20 higher concentration of methotrexate than the previous one.
0000 Q 00 coo It should be specified that the said process can be applied not only to the preparation of interleukin-2 of human origin, for which it is particularly suitable, 25 but also to the preparation of interleukin-2 of other animal origins, insofar as such molecules might be of particular interest and worthy of an industrial application, for example as a diagnostic agent or as an active principle in a drug, especially for veterinary use.
It is self-evident that this process is intended mainly to permit the preparation of glycosylated interleukin-2 as secreted by the naturally productive T lymphocytes. It should be noted that this process is also suitable for the preparation of the incompletely glycosyl2ted or unglycosylated forms of interleukin-2 i: 7 which are present in the medium after culture of the eukaryotic cells according to the invention.
The eukaryotic cells which are useful for carrying out the invention are cells of animal origin which are capable of glycosylation. Among these cells, those commonly called CHO cells by reference to their origin (Chinese hamster ovary cells) are particularly suitable.
The techniques associated with the use of these cells, whether for their propagation or their transao a fection by the vectors or else for selection of the cells which have actually been transfected, are known ooon t o o to those skilled in the art. Some of them will be o o2 described in greater detail 4n the presentation of the oo.oo 15 Examples.
o 9 o00 The vectors necessary for carrying out the invention can have a variety of forms. They can consist of all or part of a viral genome and especially of the .0 0 genome of a retrovirus or a plasmid or else a cosmid.
20 A plasmid is advantageously used.
The vectors according to the invention carry, 0000 oo with the means necessary for their expression, a DNA sequence coding for dihydrofolate reduictase and a DNA sequence coding for a hybrid precursor of interleukin-2 ooooo 25 (this precursor being symbolically designated hereafter by the notation: (ps-hGH)-interleukin-2), the signal peptide of which is that of one of the natural precursors of hGH.
The coding part corresponding to the signal peptide can have one of the sequences allowed by the degeneracy of the genetic code, it being possible for the same amino acid except methionine to be coded by 2, 3, 4 or in some cases even 6 codons. A preferred nucleotide sequence coding for the signal peptide ps-hGH is the one shown in Figure 11, which gives the corresi 1 8 ponding amino acid above each of the 26 codons.
The said DNA sequences can be included in the same expression unit. It is advantageous for each one to belong to an autonomous expression unit. The means necessary for the expression of these sequences are chosen from those which are generally used for the construction of vectors intended for the transfection of eukaryotic cells. They are preferably derived from the genome of SV 40, from which it is possible in particular to prepare DNA sequences including especially O the early promoter and/or the early polyadenylation o signal Piers (1978), Nature, 273, 113-120).
ob The construction of the vectors according to the invention requires techniques which are now well o 15 known to those skilled in the art.
So3 It should be noted that, as regards the DNA sequence coding for the precursor (ps-hGH)-interleukina 2, it is advantageous to prepare a DNA (by reference
S
o for example to the work of T. Taniguchi et al. ((1983), 20 Nature, 302, 305-310) or to that of X. Devos et l.
((1983), Nucleic Acids Res., 11, 4307-4323)) complemena tary to the messenger RNA of T lymphocytes coding for the natural precursor of interleukin-2, and then to replace the sequence coding for its signal peptide with S 25 a sequence coding for the signal peptide of one of the natural precursors of hGH.
Two preferred vectors constructed for the preparation of interleukin-2 of human origin are the plasmids pSV 726 (Figure 6) and pSV 741 (Figure 9).
They each contain an expression unit for dhfr and an expression unit for the precursor (ps-hGH)-interleukin- 2. The two plasmids differ principally in the presence, nature and position of introns in the expression units.
More precisely, the plasmid pSV 726 carries an intron downstream of the sequence coding for the precursor 9 (ps-hGH)-interleukin-2 as well as an intron downstream of the sequence coding for dhfr. The plasmid pSV 741, on the other hand, carries an intron upstream of the sequence coding for the precursor (ps-hGH)-interleukin- 2 and has no intron in the region of its expression unit for dhfr.
SAccording to yet another aspect, the invention relates to cell lines which are selected, from the a o eukaryotic cells which have incorporated a vector accor- 10 ding to the invention, by culture of the said cells in o 0 0 o° o successive media, each of which contains a higher con- Co ao centration of methotrexate than the previous one. The .ono. subcultures from one medium to the next are continued o o a 0 0 until no further amplification of the secretion of the IL-2 is observed.
Examples of how the invention is carried out o o are described below. They are of course given only by o way of illustration and in no way imply a limitation.
EXAMPLES
Two vectors according to the invention, tested under conditions of transient expression, are described below as Examples. The preparation of highly productive cell lines and then the characterization of interleukin- 2 of human origin (abbreviated hereafter to IL-2) produced are subsequently described.
I. CONSTRUCTION AND TESTING, UNDER CONDITIONS OF TRANSIENT EXPRESSION, OF TWO VECTORS: THE PLASMIDS pSV 726 AND pSV 741 1. METHODS A/ CONSTRUCTION OF THE VECTORS The construction of the vectors comprises, in particular, the isolation of DNA fragments from existing vectors with the aid of restriction enzymes, the chemical synthesis of oligonucleotides, the assembly of these various fragments if appropriate after modifica- 10 tion of their termini using an enzyme such as the DNA ligase of the bacteriophage T4, the selection of the vectors by cloning after bacterial transformation in Escherichia coli and then their purification.
It requires techniques well known to those skilled in the art.
These techniques are described in the work entitled Molecular Cloning: a Laboratory Manual by T.
o on Maniatis et al., published in 1982 by the Cold Spring 0000 1oo 0 Harbor Press, New York (USA).
0000 0 0 00 oo All the restriction enzymes necessary for the 00 o 0 construction of the vectors described below are oo0oo marketed especially by New England Biolabs (USA).
The DNA ligase of the bacteriophage T4 is 0 03 available from New England Nuclear (USA).
The construction of th, vectors is explained 0 0C 0°o,0 with the help of Figures 2 to 10, for which the 0o0 following key has been adopted: 0 0-1 0o0o 00 0
I
9 000 0° co0 0 a I X X X xI DNA sequence derived from the plasmid pBR 322 DNA sequence derived from the genome of SV DNA sequence derived from the gene coding for mouse alphaglobin DNA sequence constituting the sequence coding for the natural precursor of human interleukin-2 or its variant in which the tyrosine residue has been replaced with an alanine residue in the 2-position 1
I
HindIll amHI BamH1 1 11 DNA sequence constituting the sequence coding for the precursor (ps-hGH)-IL-2 HindIII BamHI DNA sequence coding for dhfr o 4.
o oo 00 0 0 000 oo oo o 0o a 0 o00 0 o4 oo 0 o o0 0 0 0 0 o00 0006 0 4 5 B/ PREPARATION OF A DNA SEQUENCE CODING FOR THE NATURAL PRECURSOR OF INTERLEUKIN-2 OF HUMAN
ORIGIN
A DNA complerientary to the messenger RNA coding for the precursor of IL-2, isolated from human T 10 lymphocytes, was cloned.
The resulting nucleotide sequence, in which the nucleotides are grouped together in codons above which the amino acids of the precursor corresponding thereto have been given, is included in the DNA sequence 3' strand) shown in Figure 1.
C/ USE OF THE EUKARYOTIC CELLS a. Choice The DXB11 strain of DHFR- CHO cells, selected by G. Urlaub and L. Chasin ((1980), Proc. Natl. Acad. Sci.
USA, 77, 4216-4220), was chosen.
b. Procedure for transient expression The protocol described by L. Sompayrac and K.
Danna ((1981), Proc. Natl. Acad. Sci. USA, 78, 7575) was used.
For each experiment: 5*105 cells are inoculated into a Petri dish of diameter 6 cm containing 5 ml of alpha-MEM (Gibco, USA) to which 5% of fetal calf serum (Gibco) has been added.
After 24 hours of incubation at 370C, the cells are washed with 5 ml of PBS buffer Dulbecco and M.
Vogt, J. Exp. Med., 99, (1954), 167) and then covered with 1 ml of alpha-MEM to which 0.05 mol/l of tris- (hydroxymethyl)aminomethane-HCl (or tris-HCl) of pH 7.3, 12 0 0a o oo 000000 0300 0 0 0J 00 o 0 0 oc.
0 000000 00 0 0 00 O 00 0 00 00 0 0 0.2 mg of DEAE-dextran of 500 000 daltons (Sigma, USA) and 10 pg of plasmid DNA have been added.
Incubation is carried out for 7 h at 37°C, after which the cells are washed with 5 ml of PBS buffer and then covered with 5 ml of alpha-MEM to which 2% of fetal calf serum has been added.
The cells are then incubated at 37 0 C for 4 days.
The culture medium is subsequently collected and is used for measuring the IL-2-type activity.
10 D/ MEASUREMENT OF THE IL-2-TYPE ACTIVITY The biological activity of the culture media collected as in section C/b on the proliferation of the IL-2-dependent mouse T-lymphocyte line CTLL-2 Baker et al. (1979), J. Exp. Med., 149, 173) is measured according to the colorimetric test of T. Mosmann ((1983), J. Immunol. Methods, 65, 55-63). The reference preparation described in Lymphokine Research ((1984), 4, 193-227) is used as a control.
2. THE PLASMID pSV 726 A/ CONSTRUCTION The construction of the plasmid pSV 726 starts with the plasmid pSV 700 (Figure 2).
The plasmid pSV 700 results from the assembly of 5 DNA fragments: a fragment Pvull HindIII of 342 base pairs (bp hereafter) derived from the genome of SV 40 Fiers (1978), Nature, 273, 113-120) and containing the early promoter of this virus; a fragment HindIII BaimHI of 504 bp (Figure 1) containing a DNA sequence coding for the natural precursor of IL-2 as synthesized in human lymphocytes; a fragment BamHI Ball of 305 bp derived from the gene of mouse alpha-globin Nishioka and P. Leder (1979), Cell, 18, 875-882) and containing the distal intron of this gene; 13 a fragment Hpal BamIII of 133 bp derived from the genome of SV 40 and containing the early polyadenylation signal of this virus; and a fragment BamHI PvuII of 2672 bp derived from the plasmid pBR 322 Bolivar (1977), Gene, 2, 95-113).
The nucleotide sequence AGCTTCCACAATGTACAGG located at the 5' end of the coding strand of the fragment HindIII BamI (Figure 1) is then replaced o: with the synthetic sequence AGCTTCCACCATGGCTAGG so as O 10 to give, in the region of the nucleotides enclosing o the codon ATG, a sequence conforming to the consensus ,o Q oo sequence CCACCATGG described by M. Kozak ((1984), o a Nucleic Acids Res., 12, 857-872). This gives the plasmid S pSV 703 (Figure 3).
The DNA segment between the HindIII and HgiAl restriction sites, which is located in the upstream part o o of the segment HindII BamHI (whose strand is shown in Figure 12) of the plasmid pSV 703 and contains the sequence corresponding to the modified 0t 0 0 °o 20 signal peptide (this signal peptide containing an alanine residue in the 2-position instead of the tyrosine residue because of the adoption of a sequence conforming o o. t o Kozak's consensus sequence) of the natural procursor of IL-2 and to the first amino acid of mature IL-2, is then replaced with a synthetic double-stranded oligonucleotide whose 3' coding strand is shown in Figure 11.
This synthetic sequence codes from its ninth nucleotide for the signal peptide of one of the natural precursors of hGH (the amino acid sequence of this signal peptide is given in Figure 11, each amino acid being above the corresponding codon), hereafter called the signal peptide of hGH, and for the first amino acid of mature IL-2.
The plasmid obtained is the plasmid pSV 706 Recombinant eukaryotic cells production interleukin-2, process and vectors for their preparation and process for the preparation of interleukin-2 The following statement is a full description of this invention including the best method of performing it known to us:- 14 14 (Figure Its segment HindIII BamHI, which carries the sequence coding for the precursor (ps-hGH)-IL-2, is shown in Figure 13.
Finally, the fragment between the EcoRI and EcoRV restriction sites of 185 bp of the plasmid pSV 706 is replaced with a fragment PvuII EcoRI of 2677 bp derived from the plasmid pSV2-dhfr Subramani et al. (1981), Molecular and Cellular Biology, 1, 854-864), which is deposited in the ATCC collection under the 10 reference 37146. The plasmid obtained is the plasmid o pSV 726 (Figure 6).
15 SV 40; it contains, downstream of the sequence 0a0 The plasmid pSV 726 contains: an expression unit for the precursor (ps-hGH)-IL-2; 0 t 0o f this unit has as its promoter the early promoter of SV 40; it contains, downstream of the sequence coding for the precursor of (ps-hGH)-IL-2, a sequence ooO containing the second intron of the gene of mouse o .o alpha-globin and then the early polyadenylation signal 00 0 Go 20 an expression unit for dhfr; this unit has as its promoter the early promoter of SV 40; it contains, downstream of the sequence coding for dhfr, the sequence o between the MboI sites in positions 4693 and 4083 according to the notation of W. Fiers on the genome of SV 40, the said sequence containing an intron for the t antigen of SV 40 and then the early polyaderylation signal of SV 40; this unit is contained in the fragment PvuII EcoRI derived from the plasmid pSV2dhfr.
B/ ADVANTAGES ASSOCIATED WITH THE USE OF THE PLASMID pSV 726 A comparative experiment was carried out.
The plasmids pSV 703, pSV 720 (described below) and pSV 726 were tested under conditions of transient expression (cf. methods), The IL-2-type activity of each I 11 15 0 0 .0 0" «f 0' Q 0 00 a 0 9 00 fa 0 o 'S 00 P o 0 a <aaf «r 00 0 0 0 00 C9 0£S 11 a a V e culture supernatant was measured (according to the protocol indicated above) in order to assess the IL-2 secretion level of which the cells transfected with each of the plasmids are capable.
The plasmid pSV 720 (Figure 5) is a derivative of the plasmid pSV 703. It was obtained by replacing the fragment between the EcoRI and EcoRV restriction sites of 185 bp of the plasmid pSV 703 with a fragment PvuII EcoRI of 2677 bp derived from the plasmid pSV2- 10 dhfr.
The plasmids pSV 720 and pSV 726 therefore carry the same expression unit for dhfr. They differ only in their respective DNA sequences coding for the signal peptide of the precursor of IL-2.
15 This sequence codes for a variant of the signal peptide of the natural precursor of IL-2 in the case of the plasmid pSV 720 and for the signal peptide of hGH in the case of the plasmid pSV 726.
Table 1 below shows the results of this experi- 20 ment: TABLE 1 PLASMID IL-2 ACTIVITY (U/ml) pSV 703 228 112 pSV 720 34 29 pSV 726 153 68 This Table shows the drop in secretion under conditions of transient expression associated with the introduction into a plasmid (pSV 703) of an expression unit for dhfr (plasmid pSV 720). It clearly indicates that the replacement of the sequence coding for the variant of the signal peptide of the natural precursor of IL-2 with the sequence coding for the signal peptide -7 '1
F-,
I 16 o 0 00 .o 00 0 0000 000 0 00 0 0 o 9 0 0 oo o o 0o 0000 0 00 of hGH (plasmid pSV 726) makes it possible significantly to improve the secretion level and even to achieve a level substantially equivalent to that determined for the original plasmid (plasmid pSV 703).
3. THE PLASMID pSV 741 A/ CONSTRUCTION The construction of the plasmid pSV 741 starts with the plasmid pSV 739 (Figure 7).
The plasmid pSV 739 results from the assembly 10 of 5 DNA fragments: a fragment EcoRV BglI of 777 bp derived from the genome of SV 40 and containing part of the early promoter of SV a fragment BglI PstI of 239 bp derived from the 15 plasmid pL 1 Okayama and P. Berg (1983), Molecular and Cellular Biology, 3, 280-289) and containing the part missing from the fragment EcoRV BglI of the early promoter of SV 40 and the 2 introns of the late messenger RNA 19 S of the protein VP2 and of the late 20 messenger RNA 16 S of the protein VP1 Fiers (1978), Nature, 273, 113-120); a fragment PstI BamHI of 525 bp consisting of the fragment HindIII BamHI of 521 bp of the plasmid pSV 706 (cf. Figure 13) extended by a linker PstI HindIII; this fragment contains the DNA sequence coding for the precursor (ps-hGH)-IL-2; a fragment BclI EcoRI of 988 bp derived from the genome of SV 40 and containing the early polyadenylation signal of this virus; and a fragment EcoRI PvuII of 2295 bp derived from the plasmid pBR 322.
The plasmid pSV 741 (Figure 9) is obtained by replacing the fragment BamHI EcoRI of the plasmid pSV 739 with a fragment PvuII EcoRI, itself resulting from the assembly of a fragment Bcll EcoRI of 988 bp 1' i n
I
r ii rl ij I a ii ii iL~ I uuea by the compound Known as i -2 17 derived from the genome of SV 40 and containing the early polyadenylation signal of this virus, and a fragment PvuII BglII of 1103 bp derived from the plasmid pSV2-dhfr.
The plasmid pSV 741 contains: an expression unit for the precursor (ps-hGH)-IL-2; this unit has as its promoter the early promoter of SV 40; it contains, upstream of the sequence coding for the precursor of IL-2, a sequence consisting of 0 10 two introns of SV 40 and, downstream of this sequence, ooo 000o the early polyadenylation signal of SV 40; and 00 0 0o an expression unit for dhfr; this unit contains the 0 early promoter of SV 40, a DNA sequence coding for o 0 0 dhfr and, downstream of this sequence, with no inter- 0 15 mediate intron, the early polyadenylation signal of SV B/ ADVANTAGES ASSOCIATED WITH THE USE OF THE o PLASMID pSV 741 0 00 A comparative experiment was carried out.
The plasmids pSV 739, pSV 741 and pSV 742 (cf.
below) were tested under conditions of transient expression (cf. methods).
The IL-2-type activity of each culture medium 000090 o was measured (according to the protocol indicated above) in order to assess tie IL-2 secretion level of which the cells transfected with each of the plasmids are capable.
The plasmid pSV 742 (Figure 10) was constructed from the plasmid pSV 739.
p 30 The fragment HindIII Xbal of 260 bp of the plasmid pSV 739 (Figure 13) was replaced with the fragment HindIII XbaI of 244 bp (Figure 12) of the plasmid pSV 703 (Figure The plasmiu obtained is the plasmid pSV 740 (Figure 8).
The plasmid pSV 742 was obtained by replacing Ar:r 18 the fragment EcoRI BamHI of the plasmid pSV 740 with a fragment PvuII EcoRI, itself resulting from the assembly of a fragment BglII PvuII of 1103 bp derived from the plasmid pSV2-dhfr, and a fragment EcoRI BclI of 988 bp derived from the genome of SV 40 and containing the early polyadenylation signal of this virus.
The plasmids pSV 741 and pSV 742 therefore carry the same expression unit for dhfr. They differ only in the composition of their respective DNA sequences coding for the signal peptide of the precursor of IL-2.
This sequence codes for a variant of the signal peptide of the natural precursor of IL-2 in the case of the plasmid pSV 742 and for the signal peptide of hGH in the case of the plasmid pSV 741.
Table 2 below shows the results of this experiment: TABLE 2 00 000' 0 0 o oo 0 0 00 0 o 00 a 0o 0 00 0000 00 0 400100 0 0 o o PLASMID IL-2 ACTIVITY (U/ml) pSV 740 171 pSV 741 46 4 pSV 742 7 2 This Table shows the drop in secretion under conditions of transient expression associated with the introduction into the plasmid pSV 740 of an expression unit for dhfr (plasmid pSV 742). It clearly indicates that the replacement of the sequence coding for a variant of the signal peptide of the natural precursor of IL-2 with a sequence coding for the signal peptide of hGH (plasmid pSV 741) makes it possible significantly to improve the IL-2 secretion level.
4 4: L 1. i I- I i I t x I 6 ot' o 00 o 0 0 19 II. PREPARATION OF HIGHLY PRODUCTIVE CELL LINES DHFR- CHO cells of the DXB11 strain were transfected with one or other of the plasmids pSV 726 and pSV 741.
The procedure described by F. Graham and A.
Van der Eb ((1973), Virology, 54, 536-539) was followed.
The cells are initially propagated in alpha- MEM (Gibco) to which 10% of fetal calf serum, ug/ml of gentamycin, 60 pg/ml of tylocin and 300 pg/ml of L-glutamine have been added (hereafter called nonselective medium).
After a washing phase, the cells inoculated the previous day at a rate of 0.8-10 for a Petri dish of diameter 10 cm are covered with non-selective medium and 10 pg of one of the plasmids are added in the presence of calcium phosphate but without salmon sperm DNA. Cells prepared in this way are incubated for 7 hours at 37 0
C.
The cells are then cultivated for 3 days at 20 37 0 C in alpha-MEM to which 5% of fetal calf serum has been added. When this incubation has ended, the cells are divided up, at a rate of 5"10 cells per dish, into Petri dishes containing medium which consists of minimum essential medium containing added salts and is marketed by Gibco under the reference 041-1095; the following have been added to the medium used here: Gibco dialyzed fetal calf serum (10% gentamycin Pg/ml), tylocin (50 pg/ml), L-glutamine (300 g/ml) and L-proline (150 pg/ml). Complemented in this way, this medium constitutes the selective medium referred to below.
The cells prepared in this way are incubated at 37 0 C for 2 weeks, the selective medium being renewed every 3 days. The colonies observed when this incubation has ended are in principle derived from cells which have ;i i I~~ 20 actually incorporated a plasmid. These colonies are removed, cultured again separately in selective medium and tested by measur ng the IL-2-type activity in order to be sure of their ability to produce IL-2.
It is in this way that, after transfection, 347 colonies with the plasmid pSV 726 could be isolated and were found to be positive.
The most productive colonies (35 000 to 50 000 units of IL-2/ml, measured after 4 days, starting from o 10 5 an initial population of 4-10 cells) were cultured.
The cells were subcultured successively in 4 prep ;ra- °o tions of selective medium, each of which contained a higher concentration (0.02, 0.05, 0.1 and then 0.2 uM) c, of methotrexate (amethopterin, Sigma) than the previous 15 one, in the manner described by F. Alt et al. ((1978), Journal of Biological Chemistry, 253, 1357-1570).
o'o At the end of this operating phase, several o highly productive lines could be selected.
Thus the line 109.12 transfected with the 20 plasmid pSV 726 is capable of an IL-2 secretion level 0o t of 250 000 units/ml, expressed in terms of activity, after 4 days of culture.
III. CHARACTERIZATION OF THE IL-2 SECRETED BY THE LINES The larger-scale culture of the highly productive lines described in section II provided, by treatment of the culture supernatants and after separation of the other constituents, the protein secreted by the cells in a sufficient amount to enable it to be characterized.
1. PURIFICATION OF THE IL-2 The IL-2 is purified from one liter of culture supernatant.
The supernatant is initially concentrated and subjected to a first purification by ion exchange chromatography on a column of Sepharose agarose (S Fast Flow Pharmacia Fine Chemical Sweden) which has 21 been equilibrated beforehand to pH 4.5 with 0.05 M ammonium acetate. Elution is carried out with the aid of 0.05 M ammonium acetate (pH 5.5) to which 0.05 M and then 0.5 M NaC1 are added.
The eluted fractions, which are found to be biologically active according to a measurement of their IL-2-type activity, are combined and the pool of combined fractions is subjected to high performance liquid chromatography on a reversed-phase column. The chosen support is a C -grafted silica gel. The column dimensions are 1.0 x 25.0 cm.
Elution is carried out with a linear gradient of acetonitrile varying from 5 to 100% in an aqueous solution containing 0.1% of trifluoro- S 15 acetic acid, over 80 min, at a flow rate of 4 ml/min.
The eluted fractions, which are biologically active, are combined and the pool of combined fractions a 0 0 is subjected to the same type of chromatography as above, oo f under identical conditions, especially elution con- 20 ditions, on a C 18 -grafted silica gel in a column of dimensions 2.1 x 10.0 cm.
The pool of eluted fractions collected from this chromatography, which are biologically active and, according to the results of electrophoresis on polyacrylamide gel in the presence of sodium dodecylsulfate (Laemli (1970), Nature, 277, 680-685), have an IL-2 purity of more than 95%, constitutes the material in which the IL-2 is characterized.
2. CHARACTERIZATION OF THE IL-2 BY DETERMINATION OF THE AMINO-TERMINAL SEQUENCE The samples to be treated are placed on the surface of a hexadimethrine bromide (or polybrene) filter. The filter is introduced into a protein sequencer (model 470 A marketed by Applied Biosystems (USA)) equipped with a chromatograph (model 130 A Applied 3 22 Biosystems), which analyzes in fine the phenylthiohydantoic derivatives formed.
The results of this determination are in agreement with the sequence already published for the natural product Robb et al. (1984), Proc. Natl.
Acad. Sci. USA, 81, 6486-6490).
The first ten amino acids of this sequence are as follows: S' 1 ,o Ala-Pro-Thr-Ser-Ser-Ser-Thr-Lys-Lys-Thr Alanine is the only residue detected in the N-terminal position. This provides confirmation that 0 4 0 "the precursor (ps-hGH)-IL-2 was cleaved correctly during secretion.
o° In conclusion, these Examples clearly demonstrate the value of the invention, which makes it possible to use eukaryotic cells convincingly for the production of interleukin-2 by utilizing the qualities inherent in the system of selection and/or amplification, based on transfection of the cells by a vector which 20 carries, with the means necessary for its expression, a sequence coding for dihydr ate reductas a sequence coding for dihydrofolate reductasc.
'i-
Claims (8)
1. DNA sequence, which comprises a DNA sequence coding for dihydrofolate reductase and a DNA sequence coding for a hybrid precursor of interleukin-2, the signal peptide of which is that of one of the natural precursors of human growth hormone.
2. DNA sequence according to claim 1, wherein the DNA sequence coding for the hybrid precursor is the DNA sequence coding for interleukin-2 of human origin, the signal peptide of which is that of one of the natural precursors of human growth hormone. S10 3. An expression vector which carries, with the means 0 0 necessary for their expression, a DNA sequence coding for o dihydrofolate reductase and a DNA sequence coding for a hybrid o o precursor of interLeukin-2, the signal peptide of which ir that of one of the natural precursors of human growth hormone. a 0 15 4. The vector according to claim 3, wherein the DNA sequence 0 00 coding for the hybrid precursor is the DNA sequence coding for interleukin-2 of human origin, the signal peptide of which is that of one of the natural precursors of human growth hormone. "0 o 5. The vector according to claims 3 or 4, which carries only o« 0 0. 20 one expression unit for dihydrofolate reductase and the precursor of interleukin-2.
6. The vector according to claims 3 or 4, which carries two 0 0 separate expression units, one for dihydrofolate reductase and the other for the precursor of interleukin-2. 25 7. The vector according to any one of claims 3 to 6, which 0 0 has the characteristics of one of the plasmids pSV 726 and pSV 741. 00 0 C
8. Eukaryotic cells producing interLeukin-2 which contain, with the means necessary for their expression, a DNA sequence according to claims 1 or 2.
9. Eukaryotic cells producing interleukin-2 which contain an expression vector according to anyone of claims 3 to 7. Eukaryotic cells according to claim 8 or claim 9, which are CHO cells.
11. A process for the preparation of the eukaryotic cells producing interleukin-2, according to any one of claims 8 to with 1 ml of alpha-MEM to which 0.05 mol/1 of tris- (hydroxymethyl)aminomethane-HCl (or tris-HCl) of pH 7.3, ii C, C 24 consisting in transfecting eukaryotic ceLLs with the aid of an expression vector according to anyone of claims 3 to 7 and then in seLecting the transfected cells producing interleukin-2, by culture in successive media, each of which contains a higher concentration of methotrexate than the previous one.
12. The process according to claim 11, wherein the eukaryotic cells are CHO cells.
13. A process fo, the preparation of interleukin-2 consisting in culturing eukaryotic ceLLs producing interLeukin-2 according to 10 anyone of claims 8 to Dated this 18th day of January 1989 SANOFI 15 Patent Attorneys for the Applicant F.B. RICE CO. o 00 o oo on o 0oa0 oo 0 0 00 0 0 00 o 0o 0 0 0 0 o o o o oo o o o o 0 00 o0 0 0 00 0000 0 0 0000 00 0 o 0 0o L. 1 -1 1/39 FIG]1 AGCTTCCACA TYR AEG MET GUN~ LEU LEU SER CYs ILE ALA TAC AGG ATG CAA CTC CTG TCT TGG ATT GCA 0 LEU SER LEU ALA LEU '/AL THE AsN CTA AGT CTT GCA CTT GTC ACA AAC -1 4 SEE ALA PRO THR SER SEE SEE AGT GCA CCT ACT TCA AGT TCT C 0 0 0 0 0 00 00 0 O 00 00 THR Lys Lys THR GLI, LEU GOf Lu GLU His LEU LEU LEU Asp LEU ACA AAG AAA ACA GAG GTA '1,iA CTG GAG CAT TTA CTT CTG GAT TTA GLN ME T ILE LEu AsH GLY ILE AsN AsH TYR Lys Asti PRO Lys LEU CAG ATG ATT TTG AAT GGA ATT AAT AAT TAC AAG AAT CCC AAA GTG THR AEG MET LEU THE PHE Lys PHE TYR MET PRO Lys Lys ALA THE ACC AGG ATG CTC ACA TTT AAG TTT TAG ATG CCC AAG AAG GCC ACA GLU LEu Lys His LEU (JLN Cys LEU GLu GLU GLU LEu Lys PRO LEU GAA CTG AAA CAT CTT CAG TGT CTA GAA GAA GAA CTC AAA CCT CTG, GLU GLU VAL LEU AsN LEU ALA GLN SEE Lys AsN PHE His LEu ARG GAG GAA GTG CTA AAT TTA GGT CAA AGC AAA AAC TTT GAG TTA AGA PRO AEG Asp LEU ILE SEE AsN ILE AsN VAL ILE VAL LEu GLU LEu CCC AGG GAG TTA ATG AGC AAT ATC AAC GTA ATA GTT GTG GAA CTA Lys GLY SEE GLu THE THE PHE MET Cys GLU TYR ALA Asp GLU THE AAG GGA TCT GAA AGA AGA TTC ATG TGT GAA TAT GGT GAT GAG ACA ALA THE ILE VAL GLU PHE LEU AsN AEG TEP ILE THR PHE Gys GLN GGA ACC ATT GTA GAA TTT CTG AAG AGA TGG ATT ACC TTT TGT CAA 133 Sep Ilie Ilie Ser THE LEU THE AGC ATG ATG TCA ACA GTG ACT TGA TAATTAAGTGGTTCCGACTTAAAAGATATGAG 3' 21/3 FIG 2 p0V74 Ff0.2Ec R Ru IIEco R 4 (Bo I ftm~ 3/3 FIG.3 pyull 0 00 0p00 0 0 00 o 04 0 0 0 a 00 000 0 00 o 00 0 p0 a0 0 0044 0 4 -BqIIU F-- 41/3 FIG A. -Eco RI I I o C I o ii 0 0 01 I Li 1 0 C C Pvu R Eco RY FIG.6 PVUI[ Hind~l A (Hgi Al) 00 0 a00 000 0 0 0 0 0 0 0 000 Bom HI cl 1) 3m HI) Eco RI is 613 (Pvull)-7 (Eco RY) V 44 4 4 4 0404 V I V 44 V It II 4444 V 44 4 4 4 4 o cc o ~4 44 4 4 44 @4 04 4 14 Eco RI 0 00 00 0040 4044 0 44 00 4 00 00 0 404 000440 0 0 00 4 0 00 0 04 0 00 0 0 ~0 9 00 00 4 0 44 4444 0 4 @4 4 7/9 FIG. 11 MET ALA THR GLY SER ARG THR SER LEU ATG GCT ACA GGC TCC CGG ACG TOC CTG LEU LEU ALA PHE GLY LEu LEU Cys LEU CTC CTG GCT TTT GGC CTG CTC TGC CTG PRO TRP LEU GLN GLU GLY SER ALA CCC TGG OTT CAA GAG GGC AGT GCT AGCTTACC L- s,/g FIG. 12 MET ALA ATG GCT ARG MET GLN LEU LEU SER CYs ILE ALA AGG ATG CAA CTC CIG TCT TGC ATI GCA AAGCTTCCACC Hind Ti LEU SER LEU ALA LEU VAL THR AsM CTA AGT CTT GCA CIT GTC ACA AAC -1 *1 SER ALA PRO AGI GCA A CCI HgiAI IHR SER SER SER ACT ICA AGI ICT IHR Lys Lys THR ACA AAG AAA ACA GLN LEU GLN LEu GLU His LEU LEu LEU Asp LEU CAG CIA CAA CIG GAG CAT TIA CIT CTG GAl ITA 00 0 ~00 ~0 0 0 0 0 00 00 0 04 0 0 GLN MET ILE LEu AsN GLY ILE AsM AsN TYR Lys AsM PRO Lys LEU CAG ATG All TIG AAT GGA All AAI AAT TAC AAG AAT CCC AAA CIC IHR ARG MET LEU IHR PHE Lys PHE TYR MET PRO Lys Ly's ALA IHR ACC AGG ATG CIC ACA III AAG ITT TAC AIG CCC AAG AAG GCC ACA GLU LEu Lys His LEU '3LN GAA CIG AAA CAT CIT CAG Cys LEU GLu GLU GLU LEU Lys PRO LEU TGTA CIA GAA GAA GAA CIC AAA CCI CIG XbaI GLU GLU VAL LEU AsN LEU ALA GLN SER Ly's AsN PHE His LEU ARG GAG GAA GIG CIA AAI ITA GCT CAA AGC AAA AAC III CAC ITA AGA PRO ARG Asp LEU ILE SER AsN ILE AsN VAL ILE VAL LEU GLU LEU CCC AGG GAC ITA AIC AGC AAT AIC AAC GIA AlA GTl CIG GAA CIA Lys GLY SER bLu THR IHR PHE MET Cys GLU TYR ALA Asp GLU THR AAG GGA ICT GAA ACA ACA TIC AIG IGI GAA TAT GCT GAl GAG ACA ALA THR ILE VAL GLU PHE LEU AsN ARG IRP ILE THR PHE Cys GLN GCA ACC All GIA GAA ITT CIG AAC AGA TGG All ACC III IGI CAA 133 Ser Ilie Ilie Ser THR LEU THR AGC AIC AIC ICA ACA CIG ACT IGA TAATIAAGTGCTICCCACTTAAAACATATCAG 3' 91/9 FIG.- 13' -26 MET ALA A AGTTACC ATG GCT Hi nd II GLY SER ARG THR SER L-EU LEU LFu ALA PHE GLY L-Eu LEU CYS GGC TCC CGG ACG TCC CTG CTC CTG GCT TTT GGC CTG CTC TGC LEU PRO TRP L-Eu GLN GLU GLY SER -1 I 1 ALA ALA PRO THR SER SER SER CCT ACT TCAAGT TCT CTG CCC TGG CTT CAA GAG GGC AGT GCT GCA ~4 00 9 000 0 00 0 00 0 0 000 09 0 00 0 0 00 0 0 ~0 THR Lys Lys THR GLN LEU GLN LEU ACA AAG AAA ACA CAG CTA CAA CTG GLU His LEU GAG. CAT TTA LEu LEU Asp LEU CTT CTG GAT TTA GLN MET ILE LEU AsN GLY ILE AsN AsN TYR Lys AsN PRO Lys LEU CAG AIG AlT TTG AAT GGA ATT AAT AAT TAC AAG AAT CCC AAA CTC THR ARG MET LEu THR PHE Lys PHE TYR MET PRO Lys Lys ALA THR ACC AGG ATG CTC ACA TTT AAG TTT TAC ATG CCC AAG AAG GCC ACA GLu LEu Lys His LEU GLN Cys LEu GAA CTG AAA CAT CTT CAG TGT A CTA XbaI GLu GLu GLU GAA GAA GAA LEu Lys PRO LEU CTC AAA CCT CTG 0000 9 00 00 4 444444 GLu GLU VAL LEu AsN LEU ALA GLN SER Lys AsN PHE His, LEu ARG GAG GAA GIG CTA AAI TTA GCT CAA AGC AAA AAC TTT CAC TTA AGA PRO ARG Asp LEU I LE SER AsN I LE AsN VAL I LE VAL L-EU GLU LEU CCC AGG GAC TTA ATC AGC AAT ATC AAC GIA ATA GTT CTG GAA CTA Lys GLY SER ULU THR THR PHE MET Cys GLu TY LA Asp GLu THR AAG GGA TCT GAA.ACA ACA TTC ATG TGT GAA TAT GCT GAT GAG ACA ALA THR ILE GCA ACC ATT VAL GLU PHE LEU AsH ARG TRP ILE THR PHE Cys GLN GTA GAA TT.T CIG AAC AGA TGG ATT ACC ITT IGI CAA Ser Ile Ilie Ser THR L-EU THR A6C AIC ATC TCA ACA CIG ACT IGA TAATTAAGTGCTTCCCACTTAAAACATATCAG 3'
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR8712166A FR2619719B1 (en) | 1987-09-01 | 1987-09-01 | PROCESS FOR OBTAINING INTERLEUKIN-2 FROM EUKARYOTIC CELLS, VECTORS NECESSARY FOR ITS IMPLEMENTATION AND HIGHLY PRODUCING CELL LINES |
| FR8712166 | 1987-09-01 |
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| AU2179688A AU2179688A (en) | 1989-03-02 |
| AU606453B2 true AU606453B2 (en) | 1991-02-07 |
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| AU21796/88A Ceased AU606453B2 (en) | 1987-09-01 | 1988-09-01 | Recombinant eukaryotic cells production interleukin-2, process and vectors for their preparation and process for the preparation of interleukin-2 |
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|---|---|
| EP (1) | EP0307285A1 (en) |
| JP (1) | JPH01165373A (en) |
| KR (1) | KR890005264A (en) |
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| FR (1) | FR2619719B1 (en) |
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| NO (1) | NO883883L (en) |
| NZ (1) | NZ225983A (en) |
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| ZA (1) | ZA886517B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU633099B2 (en) * | 1989-06-09 | 1993-01-21 | Novozymes Biopharma Dk A/S | Growth hormone fusion proteins |
| US7282352B2 (en) * | 2000-12-05 | 2007-10-16 | Applied Research Systems Ars Holding N.V. | Homogeneity and secretion of recombinant proteins in mammalian systems |
| US11116823B2 (en) | 2002-04-25 | 2021-09-14 | Takeda Pharmaceutical Company Limited | Treatment of α-galactosidase a deficiency |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4992367A (en) * | 1986-05-12 | 1991-02-12 | Hoffmann-La Roche Inc. | Enhanced expression of human interleukin-2 in mammalian cells |
| FR2639232A1 (en) * | 1988-10-21 | 1990-05-25 | Sanofi Sa | DRUGS CONTAINING A GLYCOSYLATED INTERLEUKIN-2 |
| US5417970A (en) * | 1988-10-21 | 1995-05-23 | Sanofi | Drugs containing a glycosylated interleukin-2 |
| EP0429586B1 (en) * | 1989-06-09 | 1995-08-02 | Gropep Pty. Ltd. | Growth hormone fusion proteins |
| US5679771A (en) * | 1990-02-13 | 1997-10-21 | Gropep Pty. Ltd. | Method for treating intestinal diseases |
| US5776746A (en) | 1996-05-01 | 1998-07-07 | Genitope Corporation | Gene amplification methods |
| IL126121A0 (en) * | 1996-05-01 | 1999-05-09 | Genitope Corp | Vaccines for treatment of lymphoma and leukemia |
| HU230275B1 (en) * | 1996-09-13 | 2015-11-30 | Shire Human Genetic Therapies, Inc | Method for producing purified human alpha galactosidase a compositions; pharmaceutical compositions comprising said purified compositions for use in treating disorders originating from alpha galactosidase a deficiency |
| US6083725A (en) | 1996-09-13 | 2000-07-04 | Transkaryotic Therapies, Inc. | Tranfected human cells expressing human α-galactosidase A protein |
| CA2309766C (en) | 1997-11-20 | 2008-09-30 | Vical Incorporated | Treatment of cancer using cytokine-expressing polynucleotides and compositions therefor |
| KR20020043448A (en) * | 2000-12-04 | 2002-06-10 | 리송알렉스인근 | Recombinant protein human interleukin-2 and manufacturing method of same |
| MA40094B1 (en) | 2014-08-06 | 2022-05-31 | Univ Miami | Interleukin-2/interleukin-2 alpha receptor fusion proteins and methods of use |
| EP3990479A4 (en) * | 2019-06-26 | 2023-08-02 | The Johns Hopkins University | Methods and materials for targeted expansion of immune effector cells |
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| EP0172619A1 (en) * | 1984-06-20 | 1986-02-26 | Takeda Chemical Industries, Ltd. | Novel transformant and use thereof |
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| US4992367A (en) * | 1986-05-12 | 1991-02-12 | Hoffmann-La Roche Inc. | Enhanced expression of human interleukin-2 in mammalian cells |
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1987
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1988
- 1988-08-19 DK DK467188A patent/DK467188A/en not_active Application Discontinuation
- 1988-08-30 PT PT88372A patent/PT88372B/en not_active IP Right Cessation
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- 1988-09-01 ZA ZA886517A patent/ZA886517B/en unknown
- 1988-09-01 AU AU21796/88A patent/AU606453B2/en not_active Ceased
- 1988-09-01 EP EP88402198A patent/EP0307285A1/en not_active Withdrawn
- 1988-09-01 KR KR1019880011309A patent/KR890005264A/en not_active Application Discontinuation
- 1988-09-01 JP JP63219590A patent/JPH01165373A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP0172619A1 (en) * | 1984-06-20 | 1986-02-26 | Takeda Chemical Industries, Ltd. | Novel transformant and use thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU633099B2 (en) * | 1989-06-09 | 1993-01-21 | Novozymes Biopharma Dk A/S | Growth hormone fusion proteins |
| US7282352B2 (en) * | 2000-12-05 | 2007-10-16 | Applied Research Systems Ars Holding N.V. | Homogeneity and secretion of recombinant proteins in mammalian systems |
| US11116823B2 (en) | 2002-04-25 | 2021-09-14 | Takeda Pharmaceutical Company Limited | Treatment of α-galactosidase a deficiency |
Also Published As
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| ZA886517B (en) | 1989-05-30 |
| FI884036A0 (en) | 1988-09-01 |
| NO883883D0 (en) | 1988-08-31 |
| PT88372A (en) | 1989-07-31 |
| KR890005264A (en) | 1989-05-13 |
| NO883883L (en) | 1989-03-02 |
| DK467188A (en) | 1989-03-02 |
| IL87622A0 (en) | 1989-01-31 |
| JPH01165373A (en) | 1989-06-29 |
| AU2179688A (en) | 1989-03-02 |
| FI884036A (en) | 1989-03-02 |
| DK467188D0 (en) | 1988-08-19 |
| NZ225983A (en) | 1990-09-26 |
| FR2619719B1 (en) | 1991-05-10 |
| EP0307285A1 (en) | 1989-03-15 |
| PT88372B (en) | 1993-04-30 |
| FR2619719A1 (en) | 1989-03-03 |
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